The present invention relates to a method and pharmaceutical composition for inhibiting viral replication in cells.
Ausubel, F. M. et al. Current Protocols in Molecular Biology, John Wiley and Sons, Media, Pa.
Chang, Y.-N., et al., J. Virology (1990) 7: 3358-3369.
Dartman, K., et al. (1986) Virology 151: 124-130.
Elion, G. B., et al. (1977) Proc. Natl. Acad. Sci USA 74:5617-5620.
Everett, R. D. et al. (1990) Nuc. Acids. Res. 18: 4579-4588.
Gottlieb, M. S., et al., N. Eng. J. Med. 305:425-3 (1981).
Grau, D. R., Visalli, R. V., Brandt, C. R. (1989) Invest. Opthalmol. Vis. Sci. 30: 2474-2480.
Grinnell, B. W., et al. (1989) J. Virol 63: 1604-1611.
Gruffat, H., et al. (1990) NAR 18:6835-43.
Hausheer, F. H. et al. (1990) Anti-Cancer Drug Des. 5: 159-167.
Helene, C. and Thuong, N. T. (1989) Genome 31: 413-421.
Hentosh, P., et al. (1992) Anal. Biochem. 201: 277-281.
Mitsuya, M., et al. (1985) Proc. Natl. Acad. Sci. USA 82: 7096-7100, USA.
Nelson, P. S., et al. (1989) Nuc. Acids Res. 17: 7179-7186.
Ono, A. et al. (1991) Biochem. 30: 9914-9921.
Poteat, H. T., et al., (1989) J. Virol. 63: 1604-1611.
Praseuth, D. (1988) Proc. Natl. Acad. Sci. USA 85: 1349-1353.
Smith, R. A., et al., xe2x80x9cRibavirin: A broad spectrum antiviral agent: In: Stapleton, T., Editor, Studies With a Broad Spectrum Antiviral Agent. International Congress and Symposium Service (London), Royal Society of Medicine, 3-23 (1986).
The challenge in developing an effective therapy and prophylaxis for viral disease is to achieve inhibition of viral processes without producing extreme side effects and preferably without inducing viral resistance. Since viral replication requires use of the cellular apparatus of the host, treating virus infection by inhibiting viral replication can be lethal to the infected host cells as well. Ideally, an anti-viral therapeutic would act selectively to inhibit or eliminate some vital part of viral processes, without affecting the host cell.
Currently, the most widely used anti-viral agents are nucleoside analogs. This class of drugs acts by disrupting viral replication, either by inhibiting enzymes required for nucleic acid processing, or by producing a defective viral genome, such as by premature termination of replication. As an example, acyclovir, a purine analog used in treating a variety of viral diseases, including herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) inhibits viral replication at several key points, including inhibition of viral thymidine kinase and DNA polymerase, and DNA strand elongation (Elion). Ribavirin, another purine analog, is the drug of choice in treating respiratory syncytial viruses (RSV) infection. This compound appears to act by reducing cellular GTP levels, blocking the action of several GTP-dependent viral processes (Smith). A drug used in the treatment of HIV infection is zidovudine (Azidothymidine; AZT), a thymidine analog which is particularly effective against human retroviruses. AZT acts with high affinity to block viral RNA-dependent DNA polymerase (reverse transcriptase); however, it also blocks human DNA-polymerase and causes chain termination (Mitsuya).
Other nucleic acid analogs include ganciclovir, vidarabine, idoxuridine, trifluridine and foscarnet (an inorganic phosphate analog). As indicated above, all of these drugs, by blocking viral replication, also have the capacity to disrupt normal host replication and/or DNA transcription processes as well.
Understanding of the mechanisms of infection and replication of viruses has led to alternate drug therapies, including attempts to block viral entry into cells, alter protein synthesis at the host ribosomes, complexation of viral DNA/RNA, and immunomodulation. Interferons are glycoproteins which have complex actions, including enhancement of certain host immune responses as well as direct antiviral activity. They act to prevent, rather than to treat established viral infection, and their use leads to undesirable problems including acute, serious discomfort, bone marrow suppression, viral resistance, and development of host immune response to the interferon.
Treatment with xe2x80x9canti-sensexe2x80x9d polymers of nucleic acids is a method in which the particular viral genome is the select target. The treatment provides a highly discriminating approach which would be expected to have minimal side-effects; its use as a therapeutic is hampered by problems of targeting, introduction into cells, and the quantity of material that would be required to block each strand produced by the virus.
Agents which bind to and interfere with host ribosomal protein synthesis will block viral replication. These include the toxin ricin, various plant proteins such as pokeweed anti-viral protein, alpha sarcin, and other low molecular weight compounds. In general, however, these compounds lack selectivity for viral processes. In the treatment of HIV, an RNA virus, specific inhibition of the unique retroviral enzyme, reverse transcriptase is a therapeutic target. Non-retroviral systems do not produce or use this enzyme.
In some instances, understanding of structural aspects of the mechanisms of replication of viruses has provided additional drug therapies. For example, certain viruses, contain a viral envelope which surrounds the viral capsid and nucleic acid. This envelope can serve as an additional target for therapeutic assault.
The present invention provides a method of inhibiting replication of a virus in infected cells. The method involves selecting a DNA fragment which contains covalently linked strands and which has 6-30 basepairs and a total size of less than about 100 basepairs. The sequence of the DNA fragment corresponds to that of a regulatory element in the virus. The DNA fragment is introduced into a cell in an amount sufficient to inhibit replication of the virus.
In a preferred embodiment, the two strands of the DNA fragment are linked in a 5xe2x80x2 to 3xe2x80x2 direction by a nucleotide sequence X1X2X3X4, where X1 is U or T, and X2 is U, T, G, A or C, X3 is C, and X4 is G (SEQ ID NO: 14); or X1 is G, and X2 is U, T, G, A or C, X3 is G or A, and S4 is A (SEQ ID NO: 15); or X1 is C, and X2 is U or T, X3 is U or T, and X4 is G (SEQ ID NO: 16), and where X1 is covalently attached to a 3xe2x80x2 terminus of the DNA and X4 is covalently attached to a 5xe2x80x2 terminus of the DNA.
In another embodiment, the method includes selecting a DNA fragment which further includes a nucleotide base containing a group-specific reactive moiety capable of covalently bonding to a protein. Preferably, such a reactive moiety is selected from the group consisting of 6-mercaptopurine, 5-bromo-deoxyuridine, 5-formyl-deoxyuridine, 5-hydroxylmethyl-deoxyuridine, 6-azopurine, a 2-halo-purine, and a 5 halo-uridine.
In yet another preferred embodiment, the method of the invention is used to inhibit replication of Herpes Simplex Virus. 1 and the DNA fragment has the sequence SEQ ID NO: 4.
In another aspect, the invention includes a pharmaceutical composition for treating a virus infection. The pharmaceutical composition includes a pharmaceutical excipient containing a DNA fragment, as characterized by covalently linked strands having a 6-30 basepair region whose sequence corresponds to that of a regulatory element in the virus. The fragment has a total size of, less than about 100 base pairs.
In a preferred embodiment, the DNA fragment contained in the pharmaceutical composition includes a nucleotide sequence X1X2 X3X4, where X1 is U or T, X2 is U, T, G, A or C, X3 is C, and X4 is G (SEQ ID NO: 14); or X1 is G, X2 is U, T, G, A or C, X3 is G or A, and X4 is A (SEQ ID NO: 15); or X1 is C, and X2 is U or T, and X3 is U or T, and X4 is G (SEQ ID NO: 16), and where, in each case, X1 is covalently attached a 3xe2x80x2 terminus of the DNA fragment and X4 is covalently attached to a 5xe2x80x2 terminus of the DNA fragment. This nucleotide sequence covalently links the two strands of the DNA fragment at one or both ends, in a 5xe2x80x2-3xe2x80x2 direction.
In another embodiment, the pharmaceutical composition further includes an antiviral agent selected from the group consisting of acyclovir, ribavirin, ganciclovir, zidovudine, vidarabine, idoxuridine, trifluridine and foscarnet.
In another preferred embodiment, the DNA fragment of the pharmaceutical composition includes the sequence SEQ ID NO: 4.